The role of gravity in the development and maintenance of bone on earth is controversial. Our current understanding is that gravitational unloading of bone results in bone loss (osteopenia) which typically progresses to osteoporosis and ultimately bone fracture. This is a significant problem that bed- ridden patients and the elderly experience. Astronauts in orbit experience significant osteopenia of approximately 1% to 3% of bone loss per month, which is equivalent to a year of bone loss for osteoporotic individuals on earth. Understanding bone response to varying levels of gravitational force is critical toward developing rational approaches in drug design or physical therapy toward mitigating osteopenia. Presently, the only means of exposing bone to a long-term lack of gravitational force is through orbital free fall aboard the ISS. These studies are expensive and logistically difficult to do aboard the ISS. An ideal method of study gravitational regulation of bone dynamics is to have a ground-based method to remove or reduce the gravitational force on bone. Creating an environment that amplifies the rate of bone loss will give us a tool to accelerate our understanding of the underlying genomic, proteomic and metabolomic mechanisms. The proposed study seeks to determine if ground-based magnetic levitation is a suitable simulation of orbital free fall for cell culture studies. Studying a cell culture rather than an entire animal allows for highly controlled and reproducible experiments. We plan to grow MC3T3 osteoblastic and primary osteoclastic cells in a bioreactor compatible with our ground-based levitation magnet and the ISS space-based microgravity environment. Our unique levitation magnet integrates a 37 C incubator integrated into the magnet bore allowing levitation for several weeks. The implementation partner for experiments aboard the ISS is BioServe Space Technologies in Boulder, CO. BioServe has a two-decade record of accomplishment of working with NASA on delivering biological experiments to and from low-earth orbit. Osteoblasts and osteoclasts flown on previous orbital missions show evidence that microgravity affects cell differentiation and gene expression. The metrics for the proposed study include cell morphology studies, microarray analysis to measure differential gene expression, ELISA and RT-PCR to identify markers of osteoblast and osteoclast differentiation, metabolomics, via H-1 NMR spectroscopy and quantification of MAPK, Twsg1 and NF-kB signaling pathways. With this suite of assays, we will determine similarities and differences in a number of metabolites and biomarkers for simulated microgravity and ISS microgravity environments. This project will help identify mechanism(s) and pathways that can be addressed to mitigate bone loss on earth.